DNA Repair

Previous studies revealed a crosstalk between intronic polyadenylation (IPA) and the DNA damage response (DDR). Indeed, genotoxic agents, including radiations and anticancer drugs (e.g., cisplatin that crosslinks DNA), regulate the ratio of IPA to last-exon transcripts in many genes. Conversely, multiple genes involved in the DDR, especially homologous recombination, are regulated at the IPA level. The U1 small nuclear RNA (snRNA) widely represses IPA, thereby enhancing full-length gene transcription. However, besides its implication in IPA regulation by ultraviolet-C radiation, little is known about U1 snRNA effects on the DDR and on cell sensitivity to genotoxic agents. Here, we show that U1 snRNA blockade using an antisense oligonucleotide (U1-AMO) in lung cancer cell lines enhances cell growth inhibition by cisplatin, through an increase in cisplatin-induced DNA damage. 3-seq analysis indicates that U1 snRNA blockade represses full-length mRNA expression of multiple genes of the nucleotide-excision repair and Fanconi anemia pathways, which are involved in the repair of cisplatin-DNA crosslinks. Our 3-seq analyses also reveal that moderate doses of U1-AMO and cisplatin upregulate the IPA:LE isoform ratio in overlapping but distinct sets of genes, and that U1-AMO prevents cisplatin effects on the IPA:LE ratio in a large subset of genes. Altogether, these data extend the crosstalk between IPA and the DDR and suggest that U1 snRNA targeting may be used to sensitize cancer cells to genotoxic agents.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

A fraction of patients treated with radiotherapy are known to be more sensitive to ionizing radiations. Skin fibroblasts from such radiosensitive individuals exhibit a higher cellular toxicity after irradiation and a delay in DNA repair. Deciphering the molecular mechanisms underlying these cellular defects is thus of major importance. We previously observed that the transcription factor NFATc2 is expressed at a reduced level in fibroblasts from radiosensitive patients. The present work aimed to elucidate the role of NFATc2 in the regulation of DNA repair, particularly the repair of radiation-induced double-strand breaks. We demonstrate an interaction of NFATc2 with the NHEJ repair protein Ku80 and observe that the NFATc2 RHD domain is necessary and sufficient for this interaction. Moreover, we show that NFATc2-Ku80 complexes are not colocalized to DNA double-strand breaks sites suggesting an involvement upstream of the DNA repair pathway. The silencing of NFATc2 impairs the NHEJ repair activities by delaying Ku70-Ku80 interaction in the early steps of this pathway. Finally, stable over-expression of NFATc2 in patients fibroblasts partially rescues their defective DNA repair phenotype, especially in the most radiosensitive cells. Altogether, our data reveal that NFATc2 is a regulator of DNA repair in skin fibroblasts and therefore a potential modulator of cellular radiosensitivity.

DNA replication achieves error rates as low as 10-9-10-11 per base pair through the combined contribution of base selection, exonuclease proofreading, and mismatch repair. Among those processes, base selection determines the initial level of accuracy and is strongly modulated by local sequence context. Existing models address this dependence either by fitting individual rate constants for each sequence context or by invoking the global template properties, neither of which derives sequence dependence from the underlying thermodynamics and kinetics of base pair formation. Here we present a mechanism for sequence-dependent base selection fidelity, built from two physical properties: nearest-neighbor stacking thermodynamics and directional kinetic asymmetry. The model fits the experimentally observed mutation spectra from three mismatch repair-deficient organisms well (r=0.74, 0.70, and 0.63), and predicts that base-selection accuracy varies non-monotonically with temperature in a sequence-dependent manner. Our model, therefore, provides a framework that connects sequence-dependent thermodynamic and kinetic effects during nucleotide incorporation to experimentally observed mutation rates.

PARK7 (DJ-1) is a redox-sensitive stress-response protein that supports cellular adaptation, but its role in post-transcriptional responses to genotoxic stress remains unclear. We investigated whether DJ-1 abundance determines the miRNA response to X-ray-induced DNA damage. Integrated mRNA-seq and small RNA-seq across DJ-1 states in HEK293 cells revealed a striking divergence in miRNA plasticity. DJ-1 depletion by siRNA produced minimal miRNA remodeling, with only 34 (6.3%) miRNAs differentially expressed after irradiation. In contrast, elevated DJ-1 markedly increased miRNA plasticity: irradiation altered [~]37% of detectable miRNAs, accounting for [~]90% of miRNA reads, and extensively redistributed the miRNA pool. DJ-1 overexpression was also associated with remodeling of the miRNA regulatory machinery, particularly components involved in miRNA sorting and stability, suggesting feedback regulation of the miRNA pool. Comparison of precursor and mature species revealed substantial uncoupling between transcription and mature miRNA abundance, implicating regulation at the levels of processing, maturation, or stability. Radiation-responsive coding genes in DJ-1-overexpressing cells were relatively depleted of miRNA binding sites, supporting preferential regulation of upstream regulatory nodes rather than the bulk transcriptome. Together, these findings identify DJ-1 as a determinant of post-transcriptional signaling plasticity, enabling dynamic remodeling of the miRNA regulatory state in response to genotoxic stress. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/744036v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@18824aorg.highwire.dtl.DTLVardef@111d8bcorg.highwire.dtl.DTLVardef@ac33b4org.highwire.dtl.DTLVardef@17698d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Poly(ADP-ribose) polymerases (PARPs) are important regulators of DNA repair and cellular stress responses in eukaryotes. Although mammalian PARPs have been extensively characterized, comparatively little is known about PARPs in pathogenic filamentous fungi. Here, we define biochemical features of MoPARP1 from the plant-pathogenic fungus Magnaporthe oryzae. Mutation of the conserved catalytic glutamate E714 abolished detectable PARylation activity, whereas PAR generated by MoPARP1 was hydrolyzed by human PARG, suggesting synthesis of polymeric ADP-ribose. Several mammalian PARP inhibitors were effective against MoPARP1 in vitro, consistent with conservation of the catalytic inhibitor-binding pocket. DNA substrates containing 5' phosphate enhanced MoPARP1 catalytic activity, while both wild-type MoPARP1 and the catalytically inactive MoPARP1 E714A bound diverse DNA substrates. Quantitative analyses revealed that structurally distinct DNA substrates exhibit similar apparent binding affinities yet yield markedly different catalytic outputs, suggesting that DNA engagement alone does not determine productive PAR synthesis. MoPARP1 lacks the canonical N-terminal zinc-finger domains of human PARP1 and instead depends on its WGR-containing region for DNA association. Domain truncation analyses showed the BRCT-WGR region supports high-affinity DNA binding, whereas the WGR-PARP region retains catalytic competence despite weaker DNA affinity. Together, these findings establish a mechanistic framework for DNA-dependent PARylation in a filamentous fungal plant pathogen.

Telomeres are highly susceptible to oxidative DNA damage, particularly 8-oxoguanine (8-oxoG), which is processed by glycosylase-initiated base excision repair (BER). OGG1 removes 8-oxoG opposite C, and MUTYH removes A misinserted opposite 8-oxoG to prevent mutations. While OGG1 has an established role in telomere protection, the contribution of MUTYH to telomere stability in cancer cells after oxidative DNA damage remains poorly understood. Using a chemoptogenetic system to induce targeted 8-oxoG lesions specifically at telomeres in HeLa cancer cells, we demonstrate that MUTYH is required to prevent telomere shortening, telomere loss, and genomic instability after chronic damage. Yet, telomere damage in MUTYH-deficient cells does not cause sustained DNA damage signaling or reduced cellular proliferation. Whole-genome sequencing further reveals enrichment of G to T transversions within telomeric repeats in MUTYH-deficient cells, consistent with increased mutagenesis due to unrepaired 8-oxoG:A mispairs. Combined loss of MUTYH and OGG1 rescues damage-induced telomere aberrations and genomic instability, implicating BER-generated single-strand break (SSB) intermediates as major contributors to telomere instability. In agreement, exo-FISH and S1-END-seq analyses reveal that repair-proficient cells rapidly accumulate SSB intermediates after damage, which are later resolved, whereas glycosylase-deficient cells exhibit SSBs at later time points. Together, these findings identify MUTYH as a critical guardian of telomere integrity during chronic oxidative stress and provide insight into how defective BER at telomeres contributes to genomic instability in cancer cells, with implications for cancers associated with MUTYH deficiency and mutations.

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

The STAG1 and STAG2 paralogs are mutually exclusive subunits of cohesin, a large protein complex that controls different aspects of genome organization. Loss-of-function mutations in STAG2 are common in a variety of cancers, and have been associated with increased sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). This provides a possible entry point for therapeutic exploitation. However, insights in the underlying mechanism and additional determinants of this sensitivity are currently lacking. Here we show that STAG2-deficient cell lines exhibit variable PARPi sensitivity. While wildtype TP53 enhances PARPi sensitivity in RPE1 cells, STAG2 depletion does not trigger p53 or affect PARPi sensitivity in RPE1-TP53wt cells. PARPi sensitivity is determined by the capacity of STAG1 to compensate for the lack of STAG2. It appears that STAG2 is not uniquely required for the PARPi response, and its loss predominantly sensitizes cells through a general defect in cohesin function. PARP trapping underlies this sensitivity and is strongly influenced by basal PARP1 expression levels and further enhanced by oncogenic signaling. Perturbations in sister chromatid cohesion also enhances PARPi sensitivity, correlating with aggravated cohesion defects and increased chromosome breakage. Mechanistically, cohesin impairment does not appear to compromise repair of nickase-induced DNA lesions, suggesting that cohesin-defective cells are particularly vulnerable to a distinct class of lesions associated with trapped PARP. Together, our findings identify residual cohesin function and PARP1 expression as key determinants of PARPi response and suggest that successful clinical application of PARP inhibitors in STAG2-mutant cancers will require biomarker-guided patient stratification rather than STAG2 mutation status alone.

Glyoxal (GO) is a small, highly reactive molecule that is produced naturally in cells during normal metabolism and can also come from processed foods and oxidative stress. Because of its high reactivity, glyoxal can modify DNA and proteins to form harmful products called advanced glycation end-products (AGEs), which have been linked to diseases such as diabetes, cancer, and aging. Although glyoxal is known to modify DNA and proteins, it is not well understood whether it can form DNA-protein crosslinks (DPCs), a type of DNA damage in which proteins become permanently attached to DNA. In this study, we investigated glyoxal induced DPC formation in HeLa cells using biochemical assays and mass spectrometry-based proteomics experiments. We observed that glyoxal exposure elevated cellular DPC formation in a concentration- and time-dependent manner. Cells with reduced SPRTN expression accumulated higher levels of DPCs, suggesting that SPRTN plays an important role in repairing glyoxal induced DNA damage. Proteomics experiments revealed 469 proteins exhibited elevated DNA association in glyoxal-treated samples, including histones and other proteins involved in chromatin organization, DNA replication, DNA repair, and gene expression. In-vitro experiments confirmed that glyoxal can directly crosslink DNA with histone proteins. Overall, this study provides the first evidence that glyoxal forms DNA-protein crosslinks in human cells. These findings provide a foundation for future studies on the chemical structure, biological effects and repair of glyoxal induced DNA-protein crosslinks and their possible role in human disease.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, with a life expectancy of less than two decades. HGPS is commonly caused by a point mutation in the LMNA gene which codes for lamin A, a vital component of the nuclear lamina. The HGPS mutation activates a cryptic splice site and leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also produced in small amounts in healthy individuals and has been implicated in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs), and alterations in DSB repair. DSB repair in mammalian cells normally occurs by either homologous recombination (HR), an accurate, templated form of repair, or by DNA end-joining (EJ), a non-templated rejoining of DNA ends. EJ is error-prone, although a portion of EJ events occurs precisely with no alteration to joined sequences. Previously, we reported that over-expression of progerin increased EJ relative to HR and decreased the precision of EJ. In our current work, we designed a novel model experimental system using derivatives of thymidine kinase (tk)-deficient mouse fibroblasts and incorporating a loss-of-function assay to further explore progerins impact on EJ. We established cell lines containing an integrated copy of a functional herpes tk gene with an embedded recognition site for endonuclease I-SceI. We examined EJ at the nucleotide level following induction of a DSB within the tk gene by expression of I-SceI and subsequent selection for cells that lost tk gene function. Comparison of EJ products recovered from cells expressing progerin versus from cells not expressing progerin revealed that progerin expression provoked larger DNA deletions associated with DSB repair as well as recovery of multiple repair products from individual cells, suggesting progerin impedes re-joining of DNA ends.

Mammalian base excision repair (BER) is the major repair pathway for correcting small DNA base lesions. BER operates via two sub-pathways: short patch (or single nucleotide) and long patch BER (SP-BER and LP-BER, respectively). In LP-BER, DNA polymerase beta (Pol{beta}) performs strand-displacement synthesis generating a 5'-flap that is removed by flap endonuclease 1 (FEN1). To determine the precise DNA-intermediate that "switches on" FEN1 activity, we analyzed the FEN1 cleavage position in DNA-intermediates generated by Pol{beta}. For simultaneous detection of Pol{beta}-catalyzed primer elongation and FEN1's cleavage products, we utilized DNA structures with fluorescent labels on the upstream and downstream primers. Our data indicate that FEN1 operates efficiently on DNAs containing one or more equilibrium nucleotides. FEN1 binds to the 3'-terminal equilibrium nucleotide of the primer and probes it for a 3'-OH group. The cleavage point is located directly opposite this 3'-terminal equilibrium nucleotide of the primer. The subsequent elimination of the equilibrium area leads to restoration of the ligatable structure, thereby limiting further strand-displacement synthesis. Since the product sets generated by the two enzymes are mirror-symmetrical, we propose that pausing of Pol{beta} permits FEN1 to join the Pol{beta}-DNA complex. Our study demonstrates that the FEN1-Pol{beta} interplay occurs via the generation and elimination of equilibrium nucleotide areas rather than through a "gap-translation" mechanism. The length of the equilibrium area depends on dNTP concentration, suggesting that the size of the LP-BER repair patches may differ between S-phase and the remainder of the cell cycle.

The RNase-H fold is an ancient protein fold found in diverse nucleases that is characterised by a conserved structural core (five {beta} strands and helices), canonical DDE/D catalytic residues and catalytic mechanism. This study focuses on the evolutionary conservation of the RNaseH-like domain in the transposase-derived THAP9 family among different taxonomic groups. Phylogenetic analysis demonstrates that the structural framework required for catalysis has been maintained over the course of evolution, while other peripheral regions have undergone lineage-specific diversification. This is exemplified by a Lys residue, which is conserved across mammals, can be structurally superposed on a catalytic Asp residue found in active transposases, but is not important for DNA integration. Further, the importance of conserved motifs (YREK and YKEFR motifs) near the THAP9 catalytic residues are confirmed biochemically by performing site directed mutagenesis followed by functional assays which demonstrate that substitution mutants have decreased ability to integrate DNA. Overall, these results emphasize that DNA integration activity is controlled by interactions with several structural elements rather than by individual catalytic residues.

Faithful inheritance of DNA methylation contributes to the memory of epigenetic states and protects against disease. While the mechanisms underlying DNA methylation maintenance at replication forks are well characterized, whether and how DNA methylation is altered or maintained at sites of DNA damage repair is still poorly understood. Here, by exploiting sequencing, imaging and proteomic approaches in mammalian cells exposed to UV radiation, we show that the majority of DNA methylation marks are maintained during UV damage repair and we dissect the molecular machinery involved in DNA methylation control. We detect the recruitment to sites of repair synthesis of the DNMT1 and DNMT3A DNA methylating enzymes, driven by the DNMT1 cofactor UHRF1 and by UV damage repair endonucleases. We also uncover a crosstalk with histone dynamics, whereby newly deposited H3.3 histones at UV damage sites promote the recruitment of DNMT1. Functionally, we reveal the importance of the DNA methylation maintenance machinery for the transcriptional response to UV damage and sustained cell proliferation. This work provides a comprehensive picture of DNA methylation control mechanisms following DNA damage, with important implications for our understanding of human diseases with an altered methylome.

Homologous recombination deficiency (HRD) can force cancer cells to rely on alternative DNA repair pathways, including microhomology-mediated end joining (MMEJ), an error-prone mechanism that can generate deletions with microhomology at repair junctions. Because such patterns may reflect DNA repair defects with clinical relevance, this study aimed to assess the biological and prognostic significance of microhomology-associated deletions and optimize their detection parameters to improve patient stratification and inform targeted therapeutic strategies, including PARP inhibition. We optimized the microhomology length threshold (Mlt parameter) using signal-to-noise ratios and survival models. We evaluated the utility of whole-exome (WES) versus whole-genome sequencing (WGS). We then investigated how Loss of Function (LoF) alterations affect microhomology-associated deletion burden and assessed their prognostic significance across an ovarian cancer (OV) cohort and a TCGA Pan-Cancer dataset. Applying an Mlt = 2 threshold maximized the precision and statistical reliability of microhomology-associated deletion identification. WGS yielded higher reproducibility, whereas WES proved insufficient for accurate estimation. Inactivation of tumor suppressor genes, including RB1 and CCDC122, was associated with increased microhomology-associated deletion burden. Importantly, higher burden correlated with extended overall survival in ovarian cancer. Furthermore, while baseline microhomology-associated deletion burden varies by tumor type, Pan-Cancer analysis identified candidate gene-level alterations, including FDX1 and PDE8B, whose LoF increased microhomology-associated deletion burden across tumors. Precise parameterization combined with WGS resolution highlights the extent to which specific gene losses affect deletion burden consistent with MMEJ-mediated repair. Identifying these genetic vulnerabilities and the resulting deletion burden may provide a candidate prognostic biomarker and foundation for personalized therapies.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

APOBEC3B (A3B) is a single-stranded DNA cytosine deaminase overexpressed in cancer, where it causes genomic DNA damage and mutations associated with tumor evolution. Enforced A3B overexpression triggers a dependency on the replication-stress response in different cellular models. However, whether endogenous A3B in cancer cells might yield a similar vulnerability is unclear. Here, we investigate how endogenous A3B expression and catalytic activity affect sensitivity to CHK1 inhibition, using two cancer cell lines, JHOC5 and U2OS. A3B-expressing cancer cells are sensitive to two chemically distinct CHK1 inhibitors, GDC-0575 and Prexasertib. CHK1 inhibitor sensitivity is reduced by A3B CRISPR knockout and restored by re-expressing wildtype A3B in knockout cells. Moreover, an endogenous A3B-E255A catalytic mutant generated by homology-directed repair phenocopies the reduced CHK1 inhibitor sensitivity of A3B-null cells, demonstrating a DNA deamination-dependent mechanism. CHK1 inhibition induces replication-associated DNA damage and cell-cycle perturbation dependent on A3B expression. As a result, A3B-expressing cells accumulate more pan-nuclear {gamma}H2AX, aberrant DNA-content profiles, and an expanded EdU-negative S-phase population, which are hallmarks of stalled DNA replication. In comparison, A3B-null and A3B-E255A cells retain defined cell-cycle distributions and are less sensitive to CHK1 inhibition. Together, these findings identify endogenous A3B-catalyzed deamination as a therapeutically actionable source of replication-associated DNA damage that renders tumor cells selectively dependent on CHK1 function. Statement of significanceAPOBEC3B causes mutations in cancer cells and simultaneously imposes DNA replication stress. This combines to sensitize tumor cells to chemical inhibitors of the DNA damage response kinase CHK1.

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Orc6 is the smallest and least evolutionarily conserved among all ORC subunits. In Drosophila, Orc6 binds tightly with the core ORC(1-5) and is required for DNA binding and replication initiation, whereas in Xenopus and human systems Orc6 loosely associates with the rest of the complex resulting in some differences for replication-associated activities. Despite these variations, Orc6 remains essential for viability in all species. In current study we analyzed specific residues within the C-terminal 11 helix that is critical for stable association of Orc6 with the ORC complex in Drosophila. Human Orc6 lacks these residues, however it possesses a strong nuclear localization signal (NLS) that is absent in Drosophilidae. We propose that this NLS drives human protein to the nucleus and compensates for weaker Orc6-ORC(1-5) interactions by increasing the nuclear concentration of Orc6 and shifting the equilibrium toward formation of the fully assembled ORC complex at the DNA.

Ewing sarcoma is an aggressive bone malignancy occurring in children, adolescents, and young adults. Most cases are caused by expression of the fusion oncoprotein EWSR1::FLI1, which contains the low complexity domain (LCD) of EWSR1 and the DNA-binding domain (DBD) of FLI1. Previous genomic studies indicate EWSR1::FLI1 accesses GGAA microsatellites in chromatin to function as a potent transcriptional regulator. Due to the technical challenges of purifying full-length EWSR1::FLI1, mechanistic studies biochemically characterizing its pioneer activities have been lacking. Here, we purified both full-length EWSR1::FLI1 and truncated DBD constructs to conduct biochemical and fluorescence-based experiments investigating interactions with different motifs in free DNA and nucleosomes. Both truncated and full-length EWSR1::FLI1 show efficient target binding in nucleosomes, and that the fourth alpha-helix in the FLI1 DBD enhances nucleosome-binding efficiency. Surprisingly, we also observe differences in both free DNA binding affinity and sequence preference between truncated and full-length proteins, though these changes are not apparent in nucleosome-binding assays. These findings reveal that EWSR1::FLI1 possesses a key pioneer factor property, efficiently targeting its binding site within nucleosomes, and that full-length EWSR1::FLI1 binding shifts to preferentially target GGAA repeats, even on motifs that bind a single EWSR1::FLI1 protein.